For many industrial processes that use starches, it is desirable to have amylolytic enzymes that can function under high temperature to rapidly breakdown starch to reduce viscosity. Examples of such enzymes are known in the art. For example, the α-amylases from Bacillus licheniformis or Bacillus stearothermophilus, AmyL and AmyS respectively, facilitate liquefaction of starches at high temperature. In the absence of added calcium, AmyL and variants of AmyL have higher thermostability than AmyS amylase, and thus are preferred for production of glucose and fructose (e.g., HFCS). AmyS and variants of AmyS are preferred in ethanol producing processes because they have higher specific activity on cornstarch at high temperature than does AmyL or its variants. Thus, AmyS has a faster initial rate of substrate viscosity reduction, which is a highly desirable attribute in starch liquefaction processes. However, AmyS has an undesirable characteristic in that its catalytic activity results in a higher final viscosity at the end of the process than that obtained with AmyL or its variants. The higher final viscosity is likely the result of the lower thermostability, i.e. AmyS is simply inactivated more quickly by the high temperature of the liquefaction process.
Methods of increasing the thermostability of enzymes have been studied. The thermostability of Amy Q (B. amyloliquefaciens amylase) was enhanced by the deletion of two amino acids, R176-Gly177, (numbering relative to amino acid sequence of AmyQ) as shown by Suzuki et al. (1989)(J. Biol. Chem. 264:18933), which are absent from the AmyL sequence. The thermostability of AmyS-type amylases can be increased by the deletion of two amino acid residues, R179-G180, (AmyS numbering) from a loop (F178 to A184) as shown by Igarashi et al. 1998 (Biochem. Biophys. Res. Comm. 248:772). However, a mutated AmyS enzyme with this deletion has a lower specific activity for corn starch hydrolysis at high-temperature than the parent enzyme, negating one of the principal advantages of AmyS amylases, as shown by Shiau et al. (2003) (Appl. Environ. Micro. 69:2383).
As discussed above, it is known in the art that, in the absence of added calcium, wild-type AmyL amylase is more thermostable than AmyS amylase. It is further known in the art that the AmyQ, an α-amylase from Bacillus amyloliquefaciens that is highly homologous to both AmyS and AmyL, is less thermostable than either AmyS or AmyL. Suzuki et al. (1989) demonstrated that for AmyL-AmyQ-derived hybrids, the N-terminal portion of the AmyL enzymes was required to obtain high stability.